Molecular transporter compositions comprising dendrimeric oligoguanidine with a tri-functional core that facilitates delivery into cells in vivo

ABSTRACT

Novel molecular transporter compositions and their use for transporting bioactive substances into cells in living animals are disclosed. To afford in vivo delivery, the composition is covalently linked to the bioactive substance and the resultant composite structure is introduced into the subject. The transporter composition includes multiple guanidine moieties on a dendrimeric scaffold having a tri-functional core. The tri-functional core is a phosphorodiamidate or phosphoramide moiety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of Ser. No. 12/079,577, filedMar. 26, 2008, by the present inventor.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the preparation and use of dendrimericoligoguanidine on a tri-functional core effective for transportingbioactive substances into the cytosol of cells in vivo. The compositionscontain guanidinium headgroups assembled around a tri-functionalphosphoramide or phosphorodiamidate core.

2. Objects and Advantages

While considerable structural diversity is found among drugs and probemolecules which act on intracellular targets, the physical properties ofmost of these agents are restricted to a narrow range to ensure passagethrough the polar extra-cellular milieu and the non-polar lipid bilayerof the cell. This can be problematic in the field of drug discoverywhere the bioactive substances are impermeable or poorly permeable tothe cells in animals. Cellular membranes are particularly impermeable tohighly charged compounds such as polynucleotides, or neutral antisensemacromolecules, such as Morpholino oligos and peptide nucleic acids.When a high molecular weight bioactive molecule (e.g. Morpholino oligo)is administered to an organism, its medicinal utility is generallylimited by its inability to efficiently gain access to its intracellulartarget.

Researchers have attempted to develop technologies for enhancing thetransport of chemical compounds across organismal barriers. Forinstance, Frankel et al. report the conjugation of selected molecules toHIV TAT protein (Frankel et al., PCT Pub. No. WO 91/09958 (1991)).Barsoum discusses the use of the HIV TAT peptide sequence RKKRRQRRR forenhancing transport across cellular membranes (Barsoum et al., PCT Pub.No. WO 94/04686 (1994)). Wender et al. discuss the use of oligoargininemoieties for increasing the delivery of various molecules acrosscellular membranes (Wender et al., US2003/0032593).

The enormous potential of arginine-based molecular transporters hasstimulated efforts to develop improved structures for delivery of largepolar molecules into cells. However, researchers have concluded from alarge number of molecular transporters containing arginines in peptideor peptoid assembly, or guanidines in a variety of backbones, that (a)the guanidine headgroups are principally responsible for its uptake intocells, (b) backbone chirality is not critical for cellular uptake, and(c) the number of guanidine head groups between 7 and 15 is optimal forefficient uptake (Rothbard, J. B., et al. J. Am. Chem. Soc.126:9506-9507 (2004)). Therefore, methods and compositions have beendescribed for transporting drugs and macromolecules across biologicalmembranes in which the drug or macromolecule is covalently attached to atransport polymer consisting of a scaffold containing oligoguanidines.

However, the practical application of such oligoguanidine transportersis generally limited due to their high cost and difficulty of use.Usually these oligoguanidines are prepared using a solid-phasesynthesizer. Although this approach is readily automated and allows forthe synthesis and purification of long oligomers, it suffers drawbacksincluding high cost, limited scalability, and the need for resinattachment and cleavage. In contrast, solution phase synthesis couldavoid the cost and scale restrictions of resins. Despite numerousreports about the importance of guanidine groups in the peptidicbackbone of oligoguanidines, most oligoguanidine delivery moieties havea linear structure, while relatively few attempts have been made usingguanidine groups in branched architectures. In one such rare case,polyguanidino dendrimers using triamine-based diamino acid monomericunits were synthesized and their delivery efficacy out-performed anoligoarginine reference standard (Wender, P. A., et al. Organic Letters7:4815-4818 (2005)). Branched-chain arginine peptides also have theability to translocate through cell membranes and to bring exogenousproteins into cells (Futaki, S., et al. Biochemistry 41:7925-7930(2002)). Dendrimeric oligoguanidines based on amino triol subunits arecapable of translocation through the cell membrane (Chung, H.-H., et al.Biopolymers (Pept. Sci) 76:83-96 (2004)). An alternative architecturebased on amino triacid scaffold demonstrated that the dendrimericmolecular transporters can not only enable transport of bioactive cargoacross the cell membrane, but also control the delivery into definedintracellular compartments (Huang, K., et al. Bioconjugate Chem. 18:403-409 (2007)).

However, the syntheses of the dendrimeric oligoguanidines mentionedabove are lengthy or involve expensive reagents for their assembly. Aneed clearly exists for new compositions and methods offering superiorperformance in transporting compounds across biological barriers, aswell as being more cost-effective to make and to link to their bioactivesubstances to be transported into cells. The present invention fulfillsthese and other needs.

SUMMARY OF THE INVENTION

Dendrimers represent an attractive transporter scaffold that offers theadvantages of economical assembly of oligoguanidine transporters througha variant of a segment-multiplying strategy. In the parent patentapplication (U.S. application Ser. No. 12/079,577 by the presentinventor), triazine was used as a tri-functional core for assembly ofoligoguanidine. In this continuation-in-part application, an alternatetri-functional phosphoramide core also possesses an orthogonal core inwhich two sites can be used for branching side arms (or side chains)while a bioactive substance can be attached to its third functional site(or a trunk, a linking group for conjugation). Specifically, each arm ofa two-arm dendrimer core can be extended with a two-arm segment to givea four-arm first generation product. Repetition of this cycle then leadsto an eight-arm system capable of incorporating eight guanidine groups.Therefore, dendrimeric assembly is one of the most efficient ways toconstruct a molecule containing multiple head groups, which in thisspecific case, will be guanidine entities. Moreover, since phosphoramidearchitecture is strange in the living system, some problems such asantigenicity and toxicity, which many peptide-based moleculartransporters suffer from, may be avoided, and therefore bioavailability,stability and safety for use in vivo may be improved.

One aspect of the invention relates to a method for the preparation of ascaffold by using phosphoramide as its core structure. One site on thistriple functional core provides for linking covalently to a bioactivesubstance via a leash or a linking group which can be either permanentor cleavable by biological means, such as proteases, lipase orreductases, or by other means such as photolysis. The other twofunctional sites of the core are used for anchoring the dendrimericarms.

Another aspect of the invention pertains to a method for the preparationof an oligoguanidine compound, comprising the steps of: (a) contactingan oligomer having a plurality of chemically tethered amines, wherein aportion of the tethered amines have attached protecting groups, with aprotecting group removal agent to remove each of the protecting groupsto produce an oligomer having a plurality of chemically tethered amines;and (b) contacting said oligomer having a plurality of chemicallytethered amines with a guanidinylation reagent to convert each of saidchemically tethered amines to a guanidinyl group to produce anoligoguanidine compound.

Yet another aspect of the invention relates to the process where thechemically tethered amines generated from removal of protecting groupsare used directly for converting to guanidine by a specific compositioncontaining a certain concentration of ammonia and a selectedguanidinylation reagent, and by selected reaction conditions including aselected range of reaction time and reaction temperature.

Yet another aspect of the invention relates to the synthetic schemewhere the protected scaffold is attached to a bioactive substance inprotected form. Removal of the protecting groups to generate thechemically tethered amines, followed by addition of a guanidinylationreagent to convert each of said chemically tethered amines to aguanidinyl group produces a conjugate containing the transportercomposition and a bioactive substance.

Yet another aspect of the invention relates to the compositionscomprising oligoguanidine delivery moiety covalently linked tosubstantially non-ionic antisense oligos including Morpholino oligos.

Yet another aspect of the invention relates to the use of saidcompositions for modifying gene expression in living subjects.Morpholinos have been shown to be effective to block translation, toalter mRNA splicing and to block binding of regulatory proteins to RNA.Splicing-blocking morpholinos can delete exons and facilitate the studyof specific splice-forms of a gene with multiple splice variants. Amorpholino oligo conjugated with the transporter composition of thisinvention works effectively in vivo to block translation of a selectedmRNA and to modify splicing of a selected pre-mRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a representative synthetic scheme for assembling anoctaguanidine of the invention. (a): The synthetic components areprepared from readily available starting materials. (b-d): The assemblystarts from phosphorus oxychloride. The first chloride is substituted bya nitrogen moiety used at a later stage for adding a linking group forconjugating with a bioactive substance. The other two chlorides aresubstituted by dialcoholamine, doubling the functional groups totetra-alcohol. Activation of the tetra-alcohol to reactive carbonate,followed by introduction of secondary amine entities each containing twosuitably protected primary amino groups, gives a phosphoramide corescaffold containing a central nitrogen in protected form and eight sidechains each with a protected primary amino group. After removal of theprotecting group of the central nitrogen, a linking group is installed,the other end of which reacts to conjugate with a bioactive substance.Removal of the protecting groups for the side chains, followed byguanidinylation, gives a conjugate of transporter composition andbioactive substance. (e): Morpholino as a representative bioactivesubstance is shown in a conjugate with the transporter composition.

FIG. 2 illustrates a synthetic scheme of assembling a protected centralnitrogen functional group and a phosphoramide scaffold containing fourside chains each with a protected primary amine.

FIG. 3 illustrates a synthetic scheme of assembling a protected centralnitrogen functional group and a phosphoramide scaffold containing sixside chains each with a protected primary amine. The multiplication ofthe side chains takes the advantage of aminotriol, where the amino groupreacts with the dichloro derivative and the resultant hexa-alcohols areactivated to form reactive carbonate. The carbonates are then treatedwith suitably protected amines to give hexa-primary amines in aprotected form.

FIG. 4 illustrates a synthetic scheme of assembling a protected centralnitrogen functional group and a phosphoramide scaffold containing twelveside chains with a protected primary amine. By using the same reactivecarbonate intermediate as shown in FIG. 3, and introducing a triaminecontaining a reactive secondary amine and two suitably protected primaryamino groups, a dozen of side chains each with a protected primary amineare furnished.

FIG. 5 illustrates a synthetic scheme of assembling a protected centralnitrogen functional group and a phosphoramide scaffold containingsixteen side chains with a protected primary amine. By using the samereactive carbonate intermediate as shown in FIG. 1 b, treatment ofdiethanolamine gives octa-alcohol. Activation of the alcohol to thecorresponding carbonate intermediate, followed by introduction of atriamine containing a reactive secondary amine and two suitablyprotected primary amino groups gives a phosphoramide scaffold containingsixteen side chains each with a protected primary amine.

FIG. 6 illustrates a synthetic scheme of assembling a rigid corescaffold containing eight side chains each with a protected primaryamine and a protected central nitrogen functional group. This schemetakes advantage of the relative reactivities of phosphorus oxychloridein such a way that mono-substituted dichloro derivative can be convertedto a diamine derivative as nucleophile to attack a di-substitutedmonochloro derivative to form a dendrimeric structure containing eightside chains in a rigid core.

FIG. 7 illustrates a synthetic scheme of assembling a flexible corescaffold containing eight side chains each with a protected primaryamine and a protected central nitrogen functional group. This schemetakes advantage of phosphorodiamidate and its analogs containing a leashwhich the terminal amine, after deprotection, can be used as nucleophileto attack its own precursor, monosubstituted dichloro derivative, toform a scaffold containing eight side chains each with a protectedprimary amine.

FIG. 8 illustrates a general scheme following FIGS. 2, 3, 4, 5, 6 and 7for subsequent manipulation. The linking site generated afterdeprotection, is coupled with a linking group. The active end of thelinking group is connected with a bioactive substance. The oligoamines,generated from removing the protecting groups, undergoes guanidinylationto give an oligoguanidine conjugated with bioactive substance.

FIG. 9 illustrates one scheme of assembling oligoguanidine in apre-formed manner. Therefore, this strategy represents the complementarymethod of installing guanidine prior to conjugation with a bioactivesubstance. The same deprotection strategy is applied for generating thecentral reactive nitrogen for installation of a linker and subsequentconjugation with a bioactive substance.

DETAILED DESCRIPTION OF THE INVENTION I. Abbreviations and Definitions

Before describing detailed embodiments of the invention, it will beuseful to set forth abbreviations and definitions that are used indescribing the invention. The definitions set forth apply only to theterms as they are used in this patent. The following description of thepreferred embodiments and examples are provided by way of explanationand illustration. As such, they are not to be viewed as limiting thescope of the invention as defined by the claims. Additionally, whenexamples are given, they are intended to be exemplary only and not to berestrictive. For example, when an example is said to “include” aspecific feature, that is intended to imply that it may have thatfeature but not that such examples are limited to those that includethat feature.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

“Tri-functional core” refers to phosphorus oxychloride or itsderivatives as the starting material where one of its reactive sites canbe used to install a linking group for ultimate conjugation with abioactive substance and the other two sites used for multiplication ofside chains. The tri-functional core forms phosphoramide, orphosphorodiamidate or its analogs as central scaffold for assembly oftransporter composition containing a linking group for conjugation witha bioactive substance and multiple side chains each containing aterminal guanidinium group or a primary amino group which is convertedto a guanidine moiety.

“Oligoguanidine compound” refers to an oligomer of subunits, eachsubunit of which contains a chemically tethered guanidine group. Aguanidine residue has the general structural characteristics of aguanidine head group, plus a tether of up to 12 methylene groups linkingthe guanidine moiety to the core of the transporter composition, wherethe core includes at least one phosphoramide or phosphorodiamidate.Accordingly, in one embodiment of the invention, one component of anoligoguanidine compound has the following sectional formula A:

wherein: m and n are integers of from 2 to 6. In the formula above, theguanidine group is illustrated as being neutral. One of skill in the artwill appreciate that the extent to which an oligoguanidine compound ischarged will depend on the environment in which it is present (includingmedium, pH, etc.) and both charged and uncharged forms are contemplatedby the present invention.

The core of the oligoguanidine transporter composition is a“tri-functional core (phosphoramide or phosphorodiamidate)” which servesas the scaffold on which the guanidine-containing components areassembled. One site on this tri-functional core is used to install alinker or a linking group for conjugation with bioactive substances. Theother two sites on the tri-functional core are used to install the sidechains of amino groups in a protected form, which are subsequentlydeprotected and converted to guanidines, preferably after coupling withbioactive substances. Accordingly, in one embodiment of the invention,the core structure of the delivery composition has the following formulaB:

Wherein: m is an integer of from 0 to 10; n is an integer of from 1 to5. L is a linker or a linking group to conjugate to a bioactivesubstance. Therefore, merger of Formula A and B represents a whole pieceof transporter composition containing oligoguanidine head groups,tri-functional core scaffold and a linker bound to or suitable forbinding to a bioactive substance.

As used herein, the term “oligoguanidine compound” refers to an oligomerof subunits, each of which contains a chemically tethered group that isa guanidine or that has been chemically transformed to generate aguanidine group. Transformation to the guanidine group can be done priorto the conjugation with a bioactive substance by using an appropriatestarting material (i.e., an oligomer having chemically built-inguanidine in a suitably protected form). Alternately, an oligoguanidinecompound can be made at the end of the synthesis by a perguanidinylationstep.

A linking moiety “L” has two termini, one that covalently attaches tothe transporter composition and one that covalently bonds to a bioactivesubstance. Examples of such groups include, without limitation,carboxylic acid, carboxylic acid derivatives, alcohols, amines andthiols. For example, one end of a dicarboxylic acid is used in attachingthe transporter composition, while the other in attaching a bioactivesubstance. The cleavable linking moiety is preferable when it is used invivo. “Cleavable” in this case refers to separation of transportercomposition from the bioactive substance. The separation is effectedthrough cleavage of a covalent bond unstable in a biologicalenvironment. For example, a linking moiety containing a disulfide bondmay be cleaved in the reducing environment within cells in the livingorganism. Or a linking moiety contains a short section of peptide, whichcan be cleaved by peptidases or proteases in the living organism. Thecleavage releases a free bioactive substance from the transportercomposition.

“Delivery” refers to an increase in amount and/or rate of transporting abioactive substance across a biological bather. The term is also meantto include altering tissue distribution, localization and release of abioactive substance or agent.

“Biological bather” refers to a physiological bather to the delivery ofa bioactive substance to its intended target site. It includes, forexample biological membranes.

“Biological membrane” refers to a lipid-containing bather that separatescells or groups of cells from extracellular space.

“Bioactive substance” refers to a therapeutic compound or a diagnosticagent, as well as lead compounds in a research and development setting.Still further the term is meant to include various probes (e.g.,oligonucleotides alone or those having attached imaging agents) andsubstances effective to alter biological processes within cells.

The term “therapeutic compound” refers, without limitation, to anycomposition that can be used to the benefit of a mammalian species. Anumber of such agents cause an observable change in the structure,function or composition of a cell upon uptake by the cell. Observablechanges include increased or decreased expression of one or more mRNAs,increased or decreased expression of one or more proteins,phosphorylation of a protein or other cell component, inhibition oractivation of an enzyme, inhibition or activation of binding betweenmembers of a binding pair, an increased rate of synthesis of ametabolite, increased or decreased cell proliferation and the like.Other agents exert therapeutic effects when present in a tissue, even inthe absence of cellular entry.

The term “diagnostic agent” refers to both diagnostic imaging agents andcontrast agents. The following are non-limiting examples of diagnosticagents: radio-labeled substances such as ⁹⁹ mTc glucoheptonate;substances used in magnetic resonance imaging such as gadolinium dopedchelation agents (e.g., Gd-DTPA); metals bound to chelating agents suchas Eu, Lu, Pr, Gd, Tc⁹⁹m, Ga⁶⁷, In¹¹¹, Y⁹⁰, Cu₆₇ and Co₅₇; and, proteinssuch as □-galactosidase, green fluorescent protein and luciferase. Otherdiagnostic agents include molecular sensors.

The term “macromolecule” refers to large molecules (MW greater than 1000daltons) of biological or synthetic origin, exemplified by, but notlimited to, peptides, proteins, oligonucleotides, polynucleotides andanalogs thereof, such as peptide nucleic acids and morpholinos.

“Small organic molecule” refers to a carbon-containing agent having amolecular weight of less than or equal to 1000 daltons.

ABBREVIATIONS

In describing and claiming the present invention, the followingabbreviations will be used in accordance with the definitions set outbelow.

-   ALT Alanine aminotransferase-   AST Aspartate aminotransferase-   BNPC Bis(4-nitrophenyl) carbonate-   DCE 1,2-dichloroethane-   DCM dichloromethane-   DIEA N,N-diisopropylethylamine-   DMI 1,3-dimethyl-2-imidazolidinone-   EDCl 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide hydrochloride-   EP Endo Porter, a peptide delivery composition (Gene Tools, LLC)-   EtOAc ethyl acetate-   HMDS hexamethyldisilazane-   HOBT 1-hydroxybenzotriazole hydrate-   L linking group-   MeCN acetonitrile-   MeOH methanol-   MW molecular weight-   PG protecting group-   RT room temperature-   TBME t-butyl methyl ether-   TEA triethylamine-   TFA trifluoroacetic acid-   TFC tri-functional core-   TFE 2,2,2-trifluoroethanol-   THF tetrahydrofuran-   TLC thin layer chromatography-   Tr trityl-   VM Vivo-Morpholino commonly referred to a conjugate of transporter    composition of this invention and a morpholino oligo.

The present invention relates to the finding that guanidine residuesprovide an enhanced transport of drugs and other agents acrossbiological membranes when the residues are part of an oligoguanidinethat provides suitable assembly of the guanidines. This is in contrastto the previously described polymers of, for example, arginine, in whichthe guanidine moieties are present on essentially all subunits of thelinear transport polymer. This is also different from the previouslydescribed dendrimers of guanidines, in that the guanidine moieties inthe present invention are assembled on a tri-functional core scaffold.Thus, the transporter oligomers of the present invention can be viewedin one group of embodiments as polymers in which guanidine residues arepresent, but spaced by dendrimeric branches such that each guanidinemoiety is in the terminal head group of side chains. Synthetically, theside chains can be selected to enhance the freedom of the arm length andadjust the lipophilicity of the transporter oligomer. Furthermore, thecentral amino group can provide a site for attachment to a linking groupwhich can conjugate with bioactive substances. More importantly,guanidine moieties are prepared from their amine precursors, which aretransformed by a single step perguanidinylation. This process takes theadvantage of avoiding using the expensive reagent as compared with theprior art which involves use of costly reagents for conversion andprotection of guanidines. The whole assembly of the transportercomposition and its joining to the bioactive compound was designed in aconcise manner which can be implemented simply and cost-effectively.

III. Transporter Composition Bioactive Substance Conjugate

As noted above, the present invention provides compositions and methodsthat enhance the transport of bioactive substances across biologicalmembranes. The compositions are represented by the structures containinga tri-functional core as scaffold, oligoguanidine as head groups of eachside chains and bioactive substances attached to a linking group.Accordingly, the invention also includes the oligoguanidine compoundsdescribed herein that are chemically tethered to a bioactive substance(which includes therapeutic agents and prodrugs thereof).

The oligoguanidine compounds can be tethered to the therapeutic agent ina variety of different ways. The therapeutic agents can be linked to atransporter composition of the invention in numerous ways, including adirect bond or by means of a linking moiety. In particular, carbamate,amide, ester, thioether, disulfide, and hydrazone linkages are generallyeasy to form and suitable for most applications. In addition, variousfunctional groups (e.g., hydroxyl, amino, halogen, etc.) can be used toattach the therapeutic agent to the transporter composition. For thosetherapeutic agents that are inactive until the attached transportercomposition is released, the linker is preferably a readily cleavablelinker, meaning that it is susceptible to enzymatic or solvent-mediatedcleavage in vivo. For this purpose, linkers containing carboxylic acidesters and disulfide bonds are preferred, where the former groups arehydrolyzed enzymatically or chemically, and the latter are severed bydisulfide exchange, e.g., in the presence of glutathione.

Therapeutic agents that benefit from the transporter composition of theinvention include both small organic molecules and macromolecules (e.g.,nucleic acids, oligonucleotides and the analogs thereof, Morpholinos andpeptide nucleic acids, polynucleotides, peptides, polypeptides, andproteins). Exemplary therapeutic agents include local and systemicanti-cancer agents, antibiotics, antisense drugs, protease inhibitors,and so forth. In addition, there are numerous releasable linkers thatcan be used with the transporter composition of the invention, which canbe cleaved by phosphatases, proteases, esterases, redox compounds,photochemical agents, nucleophilic agents, acidic compounds, and soforth. Release of the therapeutic agent can be the result of eitherenzymatic or non-enzymatic action.

Turning next to the bioactive substance, the present invention findsbroad application to essentially any therapeutic or diagnostic agent.Examples of therapeutic compounds include, without limitation, thefollowing: oligonucleotides and polynucleotides formed of DNA and RNA;oligonucleotide analogs such as phosphonates (e.g., methylphosphonates), phosphoramidates, thiophosphates, locked nucleic acids(LNA), uncharged Morpholinos and peptide nucleic acids (PNAs) or theirstructural variations containing positive or negative charges; proteinssuch as kinase C, RAF-1, p21 Ras, NF-κB and C-JUN; and, polysaccharidesand polysaccharide analogs. Diagnostic agents include both diagnosticimaging agents and contrast agents. The following are non-limitingexamples of diagnostic agents: radio-labeled substances such as ⁹⁹ mTcglucoheptonate; substances used in magnetic resonance imaging such asgadolinium doped chelation agents (e.g., Gd-DTPA); metals bound tochelating agents such as Eu, Lu, Pr, Gd, Tc⁹⁹m, Ga⁶⁷, IN¹¹¹, Y⁹⁰, Cu⁶⁷and Co⁵⁷; and proteins such as □-galactosidase, green fluorescentprotein and luciferase. Still other useful agents include dyes such as,for example, fluorescein.

In certain embodiments, the transporter composition is attached to thebioactive substance through a linking moiety. Such a linking moiety hastwo termini, one that covalently bonds to the transporter compositionand one that covalently bonds to the bioactive substance. The terminieach contain a functional group that serves as a facile point ofattachment. Examples of such groups include, without limitation,carboxylic acid, carboxylic acid derivatives, alcohols, amines andthiols. For example, suberic acid is a linking moiety having acarboxylic acid at each terminus. One terminus is used in attaching thetransporter composition, while the other in attaching the bioactivesubstance.

The linking moiety is preferably cleaved in vivo. “Cleaved” in this caserefers to separation of a linking moiety from the bioactive substance.The separation is effected through cleavage of a covalent bond. Forinstance, a linking moiety containing a disulfide bond may be cleaved inthe reducing environment in the cells of a living organism, resulting inthe separation of transporter composition from the bioactive substance.Or a linking moiety contains a short section of peptide, which can becleaved by proteases in the living organism. The cleavage releases afree bioactive substance from the transporter composition.

IV. Synthesis of Transporter Moieties and Compositions

A. General Reaction

FIG. 1 provides an illustration for the synthesis of a dendrimericoctaguanidine beginning with monoprotected piperazine 1. The protectedpiperazine 1 reacts with phosphorus oxychloride to give monosubstituteddichloro derivative. The dichloride is then treated with diethanolamineto give tetraalcohol 4. The hydroxyl groups are activated with asuitable activating reagent to provide tetracarbonate 5. Thetetracarbonate 5 is treated with a secondary amine 2 obtained fromselective protection for primary amines of a triamine to tetracarbamate6 containing octaamine in a protected form. The secondary amine ofpiperazine 7 is regenerated by deprotection, which reacts with anactivated linker 3 to give the transporter moiety 8 with an activefunctional moiety at one terminus. This active functional group 8couples with a bioactive substance to give a conjugate 9. The finalremoval of protecting groups to give octaamine 10 and perguanidinylationthereof fulfills the whole entity assembly for a transporter-enabledbioactive substance 11. More detail below is provided forperguanidinylation.

FIG. 2 illustrates a synthetic scheme of assembling a protected centralnitrogen functional group and a phosphoramide scaffold containing fourside chains each with a protected primary amine. The monosubstituteddichloro derivative is treated with a secondary amine 2 obtained fromselective protection for primary amines of a triamine to give an entitycontaining phosphoramide as scaffold and four primary amines in aprotected form 13.

FIG. 3 illustrates a synthetic scheme of assembling a protected centralnitrogen functional group and a phosphoramide scaffold containing sixside chains each with a protected primary amine. The monosubstituteddichloro derivative is treated with aminotrialcohol to give hexa-alcoholintermediate 14 which is activated to form the corresponding carbonate15. The carbonate 15 is then treated with suitably protected amines togive hexa-primary amines in a protected form 16.

FIG. 4 illustrates a synthetic scheme of assembling a protected centralnitrogen functional group and a phosphoramide scaffold containing twelveside chains with a protected primary amine. By using the same reactivecarbonate intermediate 15 as shown in FIG. 3, and introducing asecondary amine 2 obtained from selective protection for primary aminesof a triamine, a dozen of side chains each with a protected primaryamine are furnished to afford compound 17.

FIG. 5 illustrates a synthetic scheme of assembling a protected centralnitrogen functional group and a phosphoramide scaffold containingsixteen side chains with a protected primary amine. By using the samereactive carbonate intermediate 5 as shown in FIG. 1 b, treatment ofdiethanolamine gives octa-alcohol 18. Activation of the alcohol 18 tothe corresponding carbonate intermediate 19, followed by introduction ofa secondary amine 2 obtained from selective protection for primaryamines of a triamine, gives a phosphoramide scaffold 20 containingsixteen side chains each with a protected primary amine.

FIG. 6 illustrates a sequence for construction of a rigid core scaffoldof octaamines in a protected form. The monosubstituted dichloroderivative is treated with piperazine to give di-secondary amine 21.Disubstituted monochlo derivative 22, obtained from treatment ofphosphorus oxychloride with the secondary amine 2 by a controllablemanner, reacts with the di-secondary amine 21, resulting in a fullyprotected form of octaamine 23.

FIG. 7 illustrates a sequence for construction of a flexible scaffold ofoctaamines in a protected form. Reaction of monoprotected alcohol oramine 24 with phosphorus oxychloride gives monosubstituted dichloroderivative 25, which is further exposed with two equivalents ofsecondary amine to give trisubstituted phosphoramide orphosphorodiamidate or its analogs 26. Removal of the trityl groupgenerates a primary amine 27, which is again treated with themonosubstituted dichloro derivative 25 to yield a fully protected formof octaamine 28.

As can be seen from FIGS. 1 to 7, use of tri-functional core(phosphoramide or phosphorodiamidate or its analogs) as a scaffold canassemble oligoamines in an efficient manner. All these amino groups areorthogonally protected (trityl vs. trifluoroacetyl). Removal of tritylgroup provides a site for installing a linking group which can be usedfor conjugation with bioactive substance. FIG. 8 illustrates a generalscheme following FIGS. 2, 3, 4, 5, 6 and 7 for subsequent manipulation.The linking site of 30, generated after deprotection of 29, is coupledwith a linking group to afford 31. The active end of the linking groupof 31 is connected with a bioactive substance to give a conjugate 32.The oligoamine 33, generated from removing the protecting groups of 32,undergoes guanidinylation to give 34, an oligoguanidine conjugated withbioactive substance.

FIGS. 1 to 7 also show the same methodology which requires adeprotection and perguanidinylation process after conjugation withbioactive substance. FIG. 9 illustrates a sequence where guanidinegroups are built-in before the transporter composition is coupled withbioactive substance. This is very useful for those bioactive substanceswhich are vulnerable to the conditions used for perguanidinylation. Theselective protection of primary amine 35 with trifluoroacetyl groupgives free secondary amine 36 which is orthogonally protected withtrityl group to give intermediate 37. The primary amine 38 re-generatedby deprotection can be transformed to guanidine 40 in a protected form.Removal of the trityl group gives the secondary amine 41 which reactswith the carbonate intermediate 5 to furnish the octaguanidine 42 in aprotected form.

Although the transformation described in FIG. 9 is useful for somebioactive substances which are vulnerable to the conditions used forperguanidinylation, one of skill in the art will readily understand thatthis route is rather lengthy whereas post-coupling perguanidinylation isthe cost-effective way. As a matter of fact, in one embodiment of theinvention, the deprotection and perguanidinylation is carried out in asingle reaction vessel without intermediate purification, providing apractical process for efficient streamlined production of conjugatecontaining transporter composition and bioactive substance.

B. Specific Embodiments of the Methods of the Invention

Accordingly, one embodiment of the invention is a method for thepreparation of an oligoguanidine compound, comprising contacting anoligomer having chemically tethered amines, at least a portion of whichare protected, with a protecting group removal agent and aguanidinylation reagent to convert each of said protected amines to aguanidinyl group, to produce an oligoguanidine compound. Morespecifically, the method may comprise the steps of (a) assembling adendrimeric structure using a tri-functional core to install a pluralityof side chains each containing a chemically tethered amine, (b)contacting one amino group which is orthogonally protected to the aminogroups at the end of each side chain with a linking group havingreactive functional entities on each end. The linking group having theremaining reactive functional entity is conjugated with a bioactivesubstance, (c) contacting an oligomer having a plurality of chemicallytethered amines, wherein a portion of the tethered amines have attachedprotecting groups, with a protecting group removal agent to remove theprotecting groups to produce an oligomer having a plurality ofchemically tethered amines; and (d) contacting the resulting oligomerwith a guanidinylation reagent to convert each of the chemicallytethered amines to a guanidinyl group to produce an oligoguanidinecompound.

In some embodiments, the oligomer having chemically tethered amines willbe isolated and purified using methods such as ion exchangechromatography, HPLC, column chromatography and the like. This oligomer(tethered amine) compound can be isolated as a salt or in neutral form.However, in a preferred embodiment, the oligomer compound havingchemically tethered amines is not isolated, but is carried on directlyto step (d). In certain embodiments, steps (c) and (d) are carried outin the same reaction vessel. Therefore, an oligomer compound havingprotecting groups on each of the amines can be treated with a protectinggroup removal agent and subsequently a guanidinylation reagent toprovide the oligoguanidine compound in a single vessel. In oneparticularly preferred embodiment, an oligomer having trifluoroacetylprotecting groups on each of the □-amines is contacted with a protectinggroup removal agent, preferably aqueous ammonia solution, and afterwardswith a guanidinylation reagent, preferably O-methylisoureahydrochloride.

In other embodiments, the oligomer having chemically tethered amines isa dendrimeric scaffold with a tri-functional core as the center piece.In another embodiment, the branching moiety from the tri-functional coreis dialcoholamine (wherein “dialcoholamine” refers to those compoundshaving hydroxyl group at each end of the side chains and the side chainscontaining multiple methylene and other heteroatom such as O, S, B andthe like.) In one particularly preferred embodiment, diethanolamine isused for multiplication of the side chains. The nitrogen atom from thedialcoholamine connects to tri-functional core and the hydroxyl groupsfrom the alcohols develop further for a plurality of side chains. Themultiplication of side chains is preferably enabled by formation ofcarbonate intermediate, which is in turn preferably connected with asecondary amine of bis(hexamethylene)triamine wherein both primaryamines are protected with preferably trifluoroacetyl groups.

Another embodiment of the invention is a method for the preparation ofan oligoguanidine compound from a suitably protected oligoamine,comprising the steps of: (a) connecting two dialcoholamine tomonosubstituted dichloro derivative to produce a tetraalcohol; (b)activating each of the hydroxyl group of the tetraalcohol to formcarbonate intermediate; (c) treating each of the carbonate groups withdialcoholamine to generate an octahydroxyl compound and thereafteractivating each of the hydroxyl group of the octahydroxyl tooctacarbonate; (d) subjecting the carbonate compound with a secondaryamine of bis(hexamethylene)triamine wherein both primary amines areprotected with preferably trifluoroacetyl groups to give oligoamines ina protected form. When step (c) is done once, an oligomer is obtainedwhich has sixteen side chains each containing a primary amine in aprotected form. In one particularly preferred embodiment, step (c) isskipped and step (d) is conducted directly after step (b) to give anoligomer having eight side chains each containing a primary amine in aprotected form.

C. Exemplary Method of the Invention

Perguanidinylation has been described for the preparation of cationicoligonucleotides (Deglane, G. et al. ChemBioChem 7:684-692 (2006)).Perguanidinylation has now been found to have utility in the preparationof oligoguanidine transporter composition as described herein.

For example, a suitable synthesis of the guanidine octamer was desireddue to the utility of this compound as a membrane transport reagent. Inview of the perguanidinylation studies noted above, octaguanidine couldin principle be prepared from an octaamine through a late stageperguanidinylation reaction. The primary amino groups can be transformedto guanidines by final perguanidination, a step offering additionaladvantages of avoiding the use of expensive protecting groups for theguanidinium subunit if it is pre-formed otherwise.

Selective protection of a triamine having a secondary amine in themiddle or close to the middle of the chain and two primary amines oneach end of the chains can be achieved to give the free secondary amineand the protected primary amines. This strategy can make use of atriamine for connecting the secondary amine to a core scaffold and forconverting two primary amines to a couple of guanidines at the finalstage. In order to manipulate the chemistry in an orthogonally protectedmanner, base-labile trifluoroacetamide protecting group is incorporatedon the primary amine for the ultimate conversion to guanidine afterdeprotection, and acid-labile trityl protecting group is installed onthe amine for the linkage with a leash connecting with a bioactivesubstance. The requisite mono-tritylated piperazine used for startingthe construction of a tri-functional core scaffold is prepared byexposing trityl chloride with excess amount of piperazine. Aftertreatment of tritylpiperazine with phosphorus oxychloride,diethanolamine is used to doubling the functional site. The tetraalcoholthus formed is activated to give tetracarbonate by usingbis(4-nitrophenyl) carbonate. Reaction of the tetracarbonate with thesecondary amine of a triamine having the primary amines protected withtrifluoroacetyl group gives rise to the octaamine in a protected form.Removal of the trityl group is achieved by acid treatment. The freeamine thus generated is exposed to a large excess of linking reagent,suberic di(4-nitrophenyl) ester, resulting in the connection of theoctaamine with the linking moiety and yielding an active ester forsubsequent conjugation with a bioactive substance.

After conjugation of the octaamine with a bioactive substance through alinking moiety, the final deprotection and perguanidinylation can beaccomplished via a single vessel operation. Since ammonia has beenutilized to effect the deprotection of trifluoroacetamides, and also asone of the reagents in the guanidinylation of amines, a single vesseloperation was investigated. Thus, treatment of the octaamine derivativewith concentrated ammonia gives a conjugate of octaamine and thebioactive substance. Without purification, the mixture is treatedfurther with O-methylisourea hydrochloride with additional 18% ammoniasolution to give octaguanidine coupled with a bioactive substance. Theconversion of octaamine to octaguanidine by using this guanidinylationsystem is virtually quantitative and the purification can be carried outby using Oasis HLB LP extraction cartridge (Waters Corporation, Milford,Mass., US).

Significantly, eight trifluoroacetamides were removed to eight primaryamines and subsequently converted to eight guanidines under mildconditions in quantitative yield. And more significantly, theammonolytic deprotection, and subsequent perguanidinylation carried outin a single reaction vessel, without intermediate purification, enablesthe practical streamline production of conjugate containing bioactivesubstance and transporter composition. This process improvementconstitutes a very valuable and cost-effective advantage over prior artproduction procedures.

D. Protecting Groups and Protecting Removal Agents

The precise conditions and reagents or agents used in the process willdepend on the nature of the protecting groups to be kept or removed.Protecting groups selected for the protection of the chemically tetheredamine groups on the side chains are generally those groups that can bedistinguished from the other protecting groups in other portions of themolecule (e.g., the trityl group protecting the amino group for linkingthe leash with a bioactive substance). Such protecting groups are oftenreferred to as “orthogonal”. Generally, the reagents and conditions canbe employed by following the guidelines in such protecting grouptreatises as Wuts and Greene, Protective Groups in Organic Synthesis,4th ed., John Wiley & Sons, New York N.Y. (2007), and the referencescited therein.

As noted above, the method of the invention involves contacting anoligomer having a plurality of chemically tethered amines, with aprotecting group removal agent to remove the protecting groups.

In one embodiment of the invention, trifluoroacetyl group is selectedfor the protecting groups on each of the chemically tethered amines forfour critical reasons: (a) methyl or ethyl trifluoroacetate is a mildand selective reagent to protect primary amines in the presence ofsecondary amine, therefore, it is useful to keep the secondary amineintact while protecting the primary amine in a polyamine (a triamine inthis particular case) so that the secondary amine can be used to connectto reactive functional groups in the scaffold; (b) a different aminogroup is used for linking the leash for conjugation with bioactivesubstance. Acid labile trityl group is selected for this amino groupsince trifluoroacetyl group is labile towards basic conditions butstable towards acidic conditions; (c) trifluoroacetyl protecting groupson the amines can be cleaved by ammonolysis, a condition also used forremoving protecting groups in some bioactive substances. Therefore,exposure of ammonia can remove protecting groups both in the bioactivesubstance and in the oligoamine moieties. This strategy proves veryuseful in the cases where for example, coupling of the precursortransporter composition, i.e. oligoamine in a protected form, is carriedout with Morpholino antisense oligo while it is still on the synthesisresin and subsequent ammonolytic treatment not only removes all theprotecting groups on the Morpholino oligo and the oligoamine moiety, butalso cleave the conjugate of precursor transporter composition andMorpholino from the synthesis resin. This advantage of convenience beingable to cleave the conjugate from the synthesis resin and remove theprotecting groups for subsequent guanidinylation paves the way forsimple operation and economic production. (d) ammonia is also a reagentfor converting amino group to guanidine in the presence of aguanidinylation agent, O-methylisourea hydrochloride. Therefore, withoutany purification, the ammonia used for deprotection of protecting groupsin oligoamine can be carried over for the subsequent guanidinylation. Bychoosing the selected protecting groups and the selected protectinggroup removal agent, the whole production process for a conjugatecontaining a bioactive substance and transporter composition issignificantly simplified and its cost is considerably reduced incomparison to prior art methods.

E. Guanidinylation Reagents

As noted above, the method of the invention involves contacting theoligomer having a plurality of chemically tethered amines, with aguanidinylation reagent to convert each of the chemically tetheredamines to a guanidinyl group to produce an oligoguanidine compound.

Any guanidinylation reagent useful for converting an amino group to aguanidinyl group can be used in the present invention. Preferably, theguanidinylation reagent is a salt of O-methylisourea. Most preferably,the guanidinylation reagent is O-methylisourea hydrochloride. Othersuitable guanidinylation reagents are described in Bernatowicz et al.,J. Org. Chem. 57: 2497-2502 (1992).

EXAMPLES

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of synthetic organic chemistry,biochemistry and the like, which are within the skill of the art. Suchtechniques are explained fully in the literature. See, for example,Kirk-Othmer's Encyclopedia of Chemical Technology; and House's ModernSynthetic Reactions.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compositions/compound/methods of the invention. Effortshave been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some experimental error and deviationsshould, of course, be allowed for. Unless indicated otherwise, parts areparts by weight, temperature is degrees centigrade and pressure is at ornear atmospheric. All components were obtained commercially unlessotherwise indicated.

Example 1 Synthesis of N-tritylpiperazine (1)

Piperazine (107.5 g, 1.25 mole) was dissolved in DCM (500 ml). Tritylchloride (69.7 g, 0.25 mole) was added to the mixture cooled in an icebath. After addition, the mixture was kept at room temperature for 30min. The mixture was washed with water (500 ml, three times). And theorganic layer was separated and dried over sodium sulfate. The mixturewas concentrated to ca. 100 ml and added to hexane (1 liter). The solidwas removed by filtration. The filtrate was evaporated to dryness togive a white solid (ca. 100 g).

Example 2 Synthesis of bis(trifluoroacetamidohexyl)amine (2)

Bis(hexamethylene)triamine (8.62 g, 40 mmol) was dissolved inacetonitrile (120 ml). Water (0.864 ml) was added to the mixture. Ethyltrifluoroacetate (16.7 ml, 140 mmol) was added to the mixture cooled inan ice bath. After addition, the mixture was refluxed for 16 hours. Thesolvents were removed by evaporation. The residue solidified uponstoring at room temperature and was used for next step withoutpurification.

Example 3 Synthesis of di(4-nitrophenyl) suberate (3)

Suberic acid (8.71 g, 50 mmol) and 4-nitrophenol (14.61 g, 105 mmol)were dissolved in DCE (100 ml). 1,3-Diisopropylcarbodiimide (16.28 ml,104 mmol) was added to the mixture. The mixture was kept at roomtemperature for 16 hours. The solid was removed by filtration. Thefiltrate was loaded on a silica gel column (silica gel 140 g), elutingwith DCM. After removal of the solvent, a white solid was obtained (18.4g, 88%).

Example 4 Synthesis ofbis(diethanolamino)-4-tritylpiperazinophosphoramide (4)

Phosphorus oxychloride (1.84 g, 12 mmol) was dissolved in a solution ofDCM (10 ml) containing DIEA (8.8 ml). N-Tritylpiperazine (1) (3.28 g, 10mmol) dissolved in DCM (10 ml) was added to the mixture cooled in an icebath. The mixture was kept at 0° C. for 30 min. The volatile materialswere removed by evaporation. Diethanolamine (12 ml) was added to themixture. The mixture was heated at 70° C. for 16 hours. The mixture wasdissolved in EtOAc (200 ml) and washed with sodium bicarbonate (150 ml)and water (200 ml×2) and dried over sodium sulfate. After removal of thesolvent, a white solid was obtained (5.1 g, 88%).

Example 5 Synthesis ofbis[di(4-nitrophenyloxycarbonyloxyethyl)amino]-4-tritylpiperazinophosphoramide(5)

Bis(diethanolamino)-4-tritylpiperazinophosphoramide (4) (2.67 g, 4.59mmol) is dissolved in acetone (40 ml). TEA (1 ml) is added to themixture, followed by bis(4-nitrophenyl) carbonate (8.4 g, 27.54 mmol).The mixture is kept at room temperature for 48 hours. The solvent isthen removed. The product is obtained by column purification to give ayellowish solid.

Example 6 Synthesis ofbis{di[di(trifluoroacetamidohexyl)aminocarbonyloxyethyl]amino}-4-tritylpiperazinophosphoramide(6)

Bis[di(4-nitrophenyloxycarbonyloxyethyl)amino]-4-tritylpiperazinophosphoramide(5)

(1.99 g, 1.6 mmol) is dissolved in acetone (40 ml). DIEA (2.8 ml, 16mmol) is added to the mixture, followed bybis(trifluoroacetamidohexyl)amine (2) (4.16 g, 8 mmol). The reactionmixture is kept at room temperature for 16 hours. The volatile materialsare removed by evaporation. The residue is chromatographed to give theproduct 6 as an oily foam.

Example 7 Synthesis of bis{di[di(trifluoroacetamidohexyl)aminocarbonyloxyethyl]amino}-[(4-nitrophenyl)oxycarbonylhexamethylenecarbonylpiperazinyl]-phosphoramide(8)

Bis {di[di(trifluoroacetamidohexyl)aminocarbonyloxyethyl]amino}-4-tritylpiperazinophosphoramide(6) (1.0 g, 0.43 mmol) is dissolved in methanol (3.2 ml) and thesolution is mixed with 5% cyanoacetic acid in TFE (5 ml). The mixture iskept at room temperature for 10 min. The solvents are removed byevaporation. The residue is then diluted with DCM (50 ml) and washedwith saturated sodium bicarbonate (30 ml). The organic layer isseparated and dried over sodium sulfate. After removal of the solvent,the crude detritylated product 7 is dissolved in acetone (10 ml). DIEA(0.4 ml, 2.3 mmol) is added to the mixture, followed bydi(4-nitrophenyl) suberate (3) (732 mg, 1.76 mmol). The reaction mixtureis kept at 50° C. for 2 hours. The solvents are removed and the productis isolated from silica gel column chromatography to give the product 8as an oily paste.

Example 8 General Procedure for Synthesis of a Conjugate 12 Containingthe Transporter Composition and Morpholino Antisense Oligo

The precursor transporter composition 8 in DMI solution containing 5%HOBT as catalyst and adequent base such as 4-methylmorpholine ortriethylamine is incubated with Morpholino at 60° C. for 2 hours. Afterremoval of the solvent, a certain volume of concentrated ammonia isadded and the mixture is incubated at 50° C. for 5 hours. Same volume of18% ammonia is added to the mixture, followed by O-methylisoureahydrochloride. The mixture is incubated at 65° C. for 45 min. Water isadded to dilute the mixture and the product is isolated by using OasisHLB LP extraction cartridge.

Example 9 Functional Quantitative Assessment of Delivery of a ConjugateContaining Transporter Composition and Morpholino in Cultured AnimalCells

Details of the method were described (Summerton, J. E., U.S. Pat. No.7,084,248). Basically, the cytosolic delivery is assessed by aquantifiable signal proportional to the amount of cargo delivered intothe cytosol. This technology was developed by Kole and co-workers (Kang,S., et al. Biochemistry 37:6235-6239 (1998)) by using thesplice-correction system, coupled with a Morpholino antisense oligotargeted against the splicing error site. A cell line has been stablytransfected with a gene that codes for an RNA transcript that includes amutation that generates a splicing error which acts to prevent thetranslation of luciferase coded by that RNA transcript. When aproperly-targeted Morpholino antisense oligo is delivered into thecytosol/nuclear compartment of such cells, the Morpholino blocks themutant site. This leads to normal translation of the luciferase, and thelight emission from that luciferase is readily quantitated in aluminometer.

The experiments are carried out in the presence of 10% serum incomparison with Endo Porter, a peptide delivery composition (Gene Tools,LLC). After incubation at 37° C. for 24 hours, the cells are lysed andassayed for both luciferase and total cell protein. The conjugate oftransporter composition of this invention and Morpholino shows somedelivery efficacy.

Alternate parallel experiments are carried out in the presence of 100%serum in comparison with Endo Porter. The conjugate of transportercomposition of this invention and Morpholino shows greater deliveryefficacy.

Example 10 Functional Quantitative Assessment of Delivery of a ConjugateContaining Transporter Composition and Morpholino In Vivo

Kole and co-workers have developed a strain of transgenic mice carryingan expressed gene that codes for an RNA transcript that potentiallycodes for a green fluorescent protein (Sazani, P., et al. NatureBiotechnology 20:1228-1233 (2002)). That RNA transcript contains amutation that causes a splicing error which prevents expression of thegreen fluorescent protein. Contacting an appropriate Morpholinoantisense oligo with that mutant RNA transcript blocks that mutant site,thereby correcting the splicing error and generating green fluorescentprotein. Thus, the technology of visualizing green fluorescence in aspecific tissue has been used to assess cytosolic delivery into cells ofthat tissue in vivo.

The ability of the conjugate containing the transporter composition ofthis invention and Morpholino to achieve cytosolic delivery in vivo isassessed. The conjugate is administered intravenously into the mice ofthe transgenic strain. Excellent delivery are achieved.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. A composition structured for entry into cells, which may be in aliving subject, comprising: a) a phosphoramide core; b) a dendrimericstructure containing 4 to 6 guanidine groups covalently linked to a bondof the phosphoramide core; c) a dendrimeric structure containing 4 to 6guanidine groups covalently linked to a second bond of the phosphoramidecore; and d) a bioactive substance covalently linked to a third bond ofthe phosphoramide core, wherein, the composition has a structure:


2. A composition structured for entry into cells, which may be in aliving subject, comprising: a) a phosphoramide core; b) a dendrimericstructure containing 4 to 6 guanidine groups covalently linked to a bondof the phosphoramide core; c) a dendrimeric structure containing 4 to 6guanidine groups covalently linked to a second bond of the phosphoramidecore; and d) a bioactive substance covalently linked to a third bond ofthe phosphoramide core, wherein, a linkage between the phosphoramidecore and the bioactive substance is readily cleaved within a cell andhas a structure:


3. The composition of claim 1, wherein the bioactive substance is aMorpholino antisense oligo.
 4. The composition of claim 2, wherein thebioactive substance is a Morpholino antisense oligo.
 5. The compositionof claim 3, wherein the Morpholino antisense oligo is effective tomodify splicing of a selected mRNA.
 6. The composition of claim 4,wherein the Morpholino antisense oligo is effective to modify splicingof a selected mRNA.
 7. The composition of claim 5, wherein theMorpholino antisense oligo is effective to correct a splicing error inthe mRNA transcript that codes for a green fluorescent protein.